Lactobacillus iners for the enhancement of urogenital health

ABSTRACT

The present invention provides methods and compositions for administration of  Lactobacillus iners  alone or together with at least one other probiotic organism such as Bifordobacterium, or another  Lactobacillus  for reduction of the risk of urogenital infection and concomitant restoration and/or maintenance of a healthy urogenital flora. A method of treatment of vaginal infections is also contemplated.

FIELD OF THE INVENTION

The present invention provides methods and compositions foradministration of Lactobacillus iners alone or together with at leastone other probiotic organism such as Bifidobacterium, or anotherLactobacillus for reduction of the risk of urogenital infection andconcomitant restoration and/or maintenance of the desired urogenitalflora.

BACKGROUND OF THE INVENTION

The microbes that inhabit the vaginal vault, play a major role inillnesses of the host, such as sexually transmitted diseases includingHIV, cancer, urinary tract infection, bacterial vaginosis and yeastvaginitis, as well as in the maintenance of a healthy tract. Anunderstanding of the nature and functionality of these organisms hasprogressed in recent years, but it is still far from optimal. For sometime the flora of so-called ‘normal’ women of child-bearing age, wasbelieved to be dominated by Lactobacillus acidophilus and L. fermentumfollowed by L. brevis, L. jensenii, L. casei, and other species.Altschul et al. (1990) J Mol Biol 215:403-10. More recently, molecularmethods have shown L. crispatus and L. jensenii to be the most commonisolates. Altschul et al. (1990) J Mol Biol 215:403-10; Antonio et al.(1999) J Infect Dis 180:1950-6; ben Omar et al. (2000) Appl EnvironMicrobiol 66:3664-73. In only one study has a previously undescribedspecies been found in 15% of women. Antonio et al. (1999) J Infect Dis180:1950-6. The development of denaturing gradient gel electrophoresis(DGGE) has provided a tool to analyze a given population of organisms inthe host. Traditionally, studies of the urogenital microflora have beenconducted by collecting urine samples or vaginal swabs and analysing thecontents by bacteriological culture techniques or by Gram-stain analysis(Nugent et al. (1991) J Clin Microbiol 29:297-301). Identification andtracking of cultivated isolates from the vaginal tract by molecularbiological techniques has aided ecological studies, although these arelogistically demanding for use in large clinical trials (Antonio et al.(1999) J Infect Dis 180:1950-6; Reid et al. (1996) FEMS Immunol MedMicrobiol 15:236; Zhong et al. (1998) Appl Environ Microbiol64:2418-23). Also, certain microbes are difficult to cultivate from thevaginal tract due to their anaerobic nature, while others may yet remainto be cultivated (Larsen et al. (2001) Clin Infect Dis 32:69-77.However, the effectiveness of DGGE compared to conventional Gram-stainanalysis (Nugent scores) and culture has not been assessed in relationto the determination of which Lactobacillus species actually inhabit thevaginal flora of pre- and post-menopausal women. Once an identificationof species has been determined, administration of appropriateLactobacillus strains for the restoration of urogenital health, forexample, is readily accomplished.

Continuous application of certain Lactobacillus strains orally andvaginally has shown to alter the flora from one indicative of bacterialvaginosis to one that is dominated by lactobacilli and regarded asnormal. Larsen et al. (2001) Clin Infect Dis 32:69-77; Muyzer (1999)Curr Opin Microbiol 2:317-22; Muyzer et al. (1998) Antonie VanLeeuwenhoek 73:127-41; Instillation of probiotic lactobacilli can make asignificant impact on the health of women.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for theestablishment and maintenance of a healthy urogenital flora. Theinvention provides at least one Lactobacillus iners, which enhances theflora's ability to out-compete urogenital pathogens. The probioticorganisms of the present invention naturally colonize the perineum,vulva, vagina and/or urethra and establish, replenish and maintain anormal healthy flora.

In one aspect of the present invention a method is provided forestablishing a healthy urogenital flora in females throughout lifecomprising administering a therapeutically effective amount of at leastone Lactobacillus iners and a pharmaceutically acceptable carrier. In afurther aspect of the method a therapeutically effective amount of asecond probiotic organism is administered. Lactobacillus is thepreferred second probiotic organism. The second probiotic organism ispreferably selected from the group consisting of L. rhamnosus, L.acidophilus, L. fermentum, L. casei, L reuteri, L. crispatus, L.plantarum, L. paracasei, L. jensenii, L. gasseri, L. cellobiosis, L.brevis, L. delbrueckii, L. helveticus, L. salivarius, L. collinoides, L.buchneri, L. rogosae, or L. bifidum. Another probiotic organism whichcan be administered with Lactobacillus iners is Bifidobacteria. TheBifidobacterium is preferably selected from the group consisting of B.bifidum, B. breve, B. adolescentis, or B. longum.

In another aspect of the present invention a prebiotic is administeredin conjunction with the probiotic organism.

In still another aspect of the present invention an ex vivo method isprovided for establishing a healthy gastrointestinal and urogenitalflora in females comprising orally administering at least one probioticorganism isolated from said female and a pharmaceutically acceptablecarrier. In a further aspect the probiotic organisms are isolated orobtained from the patient.

In yet another aspect of the present invention a method is provided formaintaining a healthy urogenital flora in females prior to, during andafter pregnancy comprising orally administering at least one probioticorganism and a pharmaceutically acceptable carrier. In a further aspectof the method a therapeutically effective amount of a second probioticorganism is administered. Lactobacillus is a preferred first probioticorganism. The Lactobacillus is preferably selected from the groupconsisting of L. rhamnosus, L. acidophilus, L. fermentum, L. casei, L.reuten, L. crispatus, L. plantarum, L. paracasei, L. jensenii, L.gasseri, L. cellobiosis, L. brevis, L. delbrueckii, L. helveticus, L.salivanus, L. collinoides, L. buchnen, L. rogosae, or L. bifidum.Bifidobacteria is a preferred second probiotic organism. TheBifidobacterium is preferably selected from the group consisting of B.bifidum, B. breve, B. adolescentis, or B. longum.

In still another aspect of the present invention an ex vivo method isprovided for restoring healthy urogenital flora in females in needthereof comprising administering at least one Lactobacillus inersisolated from the individual and a pharmaceutically acceptable carrier.

In another aspect of the present invention, a method is provided forreducing the risk of bacterial vaginosis and bacterial vaginosispathogens comprising administering a therapeutically effective amount ofat least one Lactobacillus iners and a pharmaceutically acceptablecarrier.

In still yet another aspect of the present invention a pharmaceuticalcomposition is provided which comprises a Lactobacillus iners and apharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Illustrates DGGE of 16S rRNA gene PCR amplicons of vaginalsamples from nineteen subjects (day 0, pre-study samples). Arrowindicates those sequenced in certain lanes, in unmarked lanes, alldetected fragments sequenced. BLAST sequence homologies in Table 1.

FIG. 2 Illustrates DGGE profiles of vaginal flora from five women duringstudy. L=Lactobacillus known isolates a) L. fermentum RC-14 and b) L.rhamnosus GR-1. Lanes 1-5, amplicons from samples taken at day 0(pre-study), 3, 7, 14 and 21 days after instillation of capsulescontaining lactobacilli. Annotation arrows represent DNA fragmentssequenced. Presumptive identification based on closest BLAST homologiesas follows: Subject 263, lane 1, L. delbrueckii. Subject 265, lane 1, L.crispatus, lane 3, Pseudomonas sp. Subject 260, lane 1, L. iners andlane 2, L. crispatus. Subject 261, lane 1 (top to bottom), L. iners,Arthrobacter sp. and G. vaginalis, lane 2, Pseudomonas sp. lane 3, S.agalactiae, lane 4, L. iners (all). Subject 268, lane 1 (top to bottom),Butyrivibro fibrisolvens and G. vaginalis.

FIG. 3 Illustrates lactobacilli RAPD profiles. Lanes 1 and 14, molecularweight marker (100 bp), 2 and 3, Lactobacillus strains GR-1, and RC-14,respectively, lanes 4-13, lactobacilli isolates from Subject 266(prestudy sample).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for establishingand maintaining a healthy urogenital flora in women throughout the lifecycle comprising the administration of at least one probiotic organismsuch as Lactobacillus iners and/or a second Lactobacillus and/orBifidobacterium and/or a prebiotic compound.

By “probiotic” is meant an organism which has one or more of thefollowing characteristics, an ability to: improve urogenital healththrough colonizing the gastrointestinal, vaginal or uroepithelial cellsby electrostatic, hydrophobic or specific adhesins including a collagenbinding protein; pass through the stomach and reach the small and largeintestine; grow and persist in the gastrointestinal and urogenitaltracts; inhibit the adhesion of gastrointestinal and/or urogenitalpathogens including organisms which cause urinary tract infection,bacterial vaginosis and yeast vaginitis; and other infections includingthose caused by viruses; produce acid and other substances such ashydrogen peroxide and/or bacteriocins and bacteriocin-like compoundswhich inhibit pathogen growth; produce biosurfactant or relatedby-products of growth which interfere with adhesion of pathogens tocells and materials; resist antimicrobial agents, such as nonoxynol-9spermicide; and/or enhance the host's immune function to furthermaintain a healthy urogenital flora. A preferred probiotic organism isLactobacillus iners and extracts or by-products thereof such as proteinsor peptides or amino acids.

The preferred Lactobacillus iners strains within the scope of thisinvention are aerobic, microaerophilic and strictly anaerobic isolates.A most preferred Lactobacillus species is Lactobacillus iners Y16329.Another preferred Lactobacillus species is L. iners CCP-1. Anotherpreferred Lactobacillus species is L. rhamnosus GR-1. Still anotherpreferred Lactobacillus species is L. fermentum B-54. Yet anotherpreferred Lactobacillus is L. acidophilus RC-14.

By “prebiotic” is meant a nonmetabolized, nonabsorbed substrate that isuseful for the host which selectively enhances the growth and/or themetabolic activity of a bacterium or a group of bacteria. A prebioticalso includes a nutrient utilized by lactobacilli or bifidobacteria tostimulate and/or enhance growth of lactobacilli or bifidobacteriarelative to pathogenic bacteria.

Also defined within the present invention are compositions suitable forestablishing, maintaining or restoring a healthy urogenital flora infemales throughout life which comprise one or more Lactobacillus inersviable whole cells, non-viable whole cells or cell wall fragments and apharmaceutically acceptable carrier. By “throughout life” is meant inthe neonatal period, during childhood and in the pre-menopausal andpost-menopausal periods. By “healthy urogenital flora” is meant florathat is predominantly colonized by non-pathogenic organisms and wherethere are no signs or symptoms of infection or disease.

In a preferred aspect, the Lactobacillus is aerobically,microaerophilically or anaerobically grown and is a Lactobacillus inersspecies or taxonomically related species. In a most preferred aspect,the Lactobacillus iners is L. iners Y16329. In another preferred aspect,the second Lactobacillus is aerobically, microaerophilically oranaerobically grown and is selected from the group consisting ofLactobacillus casei, L. acidophilus, L. plantarum, L. fermentum, L.brevis, L. jensenii, L. crispatus, L. rhamnosus, L. reuteri, L.paracasei, L. gassed, L. cellobiosis, L. delbrueckii, L. helveticus, L.salivarius, L. collinoides, L. buchneri, L. rogosae and L. bifidium.

The second Lactobacillus may be microaerophilically or anaerobicallygrown and selected from the group consisting of Lactobacillus rhamnosus(GR-1 (ATCC 55826), L. rhamnosus GR-2 (ATCC 55915), L. rhamnosus GR-3(ATCC 55917), L. rhamnosus GR4 (ATCC 55916), L. rhamnosus RC-9, L.rhamnosus RC-17 (ATCC 55825), L. casei var alactosus RC-21, L. casei NRC430, L. casei ATCC 7469, L. rhamnosus 81, L. rhamnosus 76, L. rhamnosus36W, L. rhamnosus 36g, L. casei RC-65, L. casei RC-15, L. casei 558, L.casei, RC-21, L. casei 55, L. casei 8, L. casei 43, L. plantarum RC-12(ATCC 55895), L. acidophilus RC-25, L. plantarum RC-19, L. jenseniiRC-11 (ATCC 55901), L. acidophilus ATCC 4357, L. acidophilus 2099 B, L.acidophilus 2155C, L. acidophilus T-13, L. acidophilus 1807B, L.acidophilus RC-16, L. acidophilus RC-26, L. acidophilus RC-10, L.acidophilus RC-24, L. acidophilus RC-13, L. acidophilus RC-14, L.acidophilus RC-12, L. acidophilus RC-22, L. acidophilus 2099B, L.acidophilus 2155C, L. acidophilus T-13, L. plantarum ATCC 8014, L.plantarum UH 2153, L. plantarum 260, L. plantarum RC-20, L. plantarum75, L. plantarum RC-6, L. fermentum A-60, L. fermentum B-54, B. longum1B, B. breve 2B, B. adolescentis 3B, B. bifidum 4B (identical ribotypeto RC-14) (ATCC 55920), L. cellobiosis RC-2, L. crispatus 1350B and L.crispatus 2142B.

In a further embodiment, the present invention describes a method ofadministering at least one Lactobacillus iners and optionally one ormore probiotic organisms for restoring a healthy urogenital flora overthe various life cycle stages of women including pregnancy andpost-menopause, wherein the flora is dominated by Mobiluncus,Gardnerella, Bacteroides, Fusobacterium, Prevotella, Peptostreptococcus,Porphyromonas, Mycoplasma or group B streptococci, or Escherichia coli,Staphylococcus sp., Enterococcus sp, Klebsiella sp, Pseudomonas sp,Streptococcus sp, Proteus sp, and other Gram negative (such ascoliforms) and Gram positive pathogens which cause urinary tractinfections and yeast including Candida albicans, for example, or whereHIV, papiloma or herpes viruses are present.

In accordance with the present invention, the Lactobacillus inersspecies will produce substances active against urogenital pathogensincluding those that cause urinary tract infections, bacterial vaginosisand yeast vaginitis such as Mobiluncus, Gardnerella, Bacteroides,Fusobacterium, Prevotella, Peptostreptococcus, Porphyromonas, Mycoplasmaof group B streptococci, or Escherichia coli, Enterococcus sp,Klebsiella sp, Pseudomonas sp, Streptococcus sp, Proteus sp, yeast, andviruses.

The Lactobacillus iners of the present invention will inhibit growthand/or adhesion of enteric pathogens to gastrointestinal surfacesincluding those that cause enteric infections. Such inhibition ofenteric pathogens is at least partly due to the production ofbiosurfactants active against such pathogens including, salmonella,shigella, listeria, campylobacter and clostridium, for example. The L.iners of the present invention will inhibit and/or prevent infectionscaused by a variety of pathogens including those described above andyeasts and viruses, including HIV.

Although this invention is not intended to be limited to any particularmode of application, oral or vaginal administration of the compositionsare preferred. One probiotic organism may be administered alone or inconjunction with a second or third different probiotic organism. By “inconjunction with” is meant together, substantially simultaneously orsequentially. The compositions may be administered in the form oftablet, pill, capsule, suppository, cream, paste, gel, ointment, doucheor liniment, for example. One preferred form of application involves thepreparation of a freeze-dried capsule comprising the composition of thepresent invention. It has been found that a capsule comprising about108-1010 Lactobacillus iners organisms is suitable. In accordance withthe present invention a capsule may contain one single or two or moredifferent species of probiotic organism(s).

By “therapeutically effective amount” as used herein is meant an amountof probiotic organism, e.g., Lactobacillus iners, high enough tosignificantly positively modify the condition to be treated but lowenough to avoid serious side effects (at a reasonable benefit/riskratio), within the scope of sound medical judgment. A therapeuticallyeffective amount of Lactobacillus iners will vary with the particulargoal to be achieved, the age and physical condition of the patient beingtreated, the severity of the underlying disease, the duration oftreatment, the nature of concurrent therapy and the specificLactobacillus iners strain employed. For example, a therapeuticallyeffective amount of Lactobacillus iners administered to a child or aneonate will be reduced proportionately in accordance with sound medicaljudgment. The effective amount of Lactobacillus iners will thus be theminimum amount which will provide the desired attachment to epithelialcells. For example, the presence of 5×109 bacteria, as viable ornon-viable whole cells, in 0.05 ml solution of phosphate buffered salinesolution, or in 0.05 ml of suspension of agar, or the dry weightequivalent of cell wall fragments, is effective when administered inquantities of from about 0.05 ml to about 20 ml.

A decided practical advantage is that the probiotic organism, e.g.Lactobacillus iners, may be administered in a convenient manner such asby the oral, intravenous (where non-viable), or suppository (vaginal orrectal) routes. Depending on the route of administration, the activeingredients which comprise probiotic organisms may be required to becoated in a material to protect said organisms from the action ofenzymes, acids and other natural conditions which may inactivate saidorganisms. In order to administer probiotic organisms such asLactobacillus iners by other than parenteral administration, they shouldbe coated by, or administered with, a material to prevent inactivation.For example, probiotic organisms may be co-administered with enzymeinhibitors or in liposomes. Enzyme inhibitors include pancreatic trypsininhibitor, diisopropylfluorophosphate (DFP) and trasylol. Liposomesinclude water-in-oil-in-water P40 emulsions as well as conventional andspecifically designed liposomes which transport lactobacilli or theirby-products to the urogenital surface.

The probiotic organisms of the invention may also be administeredparenterally or intraperitoneally. Dispersions can also be prepared, forexample, in glycerol, liquid polyethylene glycols, and mixtures thereof,and in oils.

When the probiotic organisms are suitably protected as described above,the active compound may be orally administered, for example, with aninert diluent or with an assimilable edible carrier, or it may beenclosed in hard or soft shell gelatin capsule, or it may be compressedinto tablets designed to pass through the stomach (i.e., entericcoated), or it may be incorporated directly with the food of the diet.For oral therapeutic administration, the probiotic organisms may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Compositions or preparations according to the presentinvention are prepared so that an oral dosage unit form contains about1×109 viable or non-viable e.g., lactobacilli per ml.

The tablets, pills, capsules, and the like, as described above, may alsocontain the following: a binder such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acid,and the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin may be added or a flavoringagent such as peppermint, oil or wintergreen or cherry flavoring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills or capsules may be coatedwith shellac, sugar or both.

A syrup or elixir may contain the active compound, sucrose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, theprobiotic organism may be incorporated into sustained-releasepreparations and formulations.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of the probiotic organismscalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. The specification for the noveldosage unit forms of the invention are dictated by and directlydepending on (a) the unique characteristics of the probiotic organismand the particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such probiotic for theestablishment and maintenance of a healthy urogenital flora.

The probiotic organism is compounded for convenient and effectiveadministration in effective amounts with a suitable pharmaceutically orfood acceptable carrier in dosage unit form as hereinbefore disclosed. Aunit dosage form can, for example, contain the principal active compoundin an amount approximating 109 viable or non-viable, e.g., lactobacilli,per ml. In the case of compositions containing supplementary ingredientssuch as prebiotics, the dosages are determined by reference to the usualdose and manner of administration of the said ingredients.

By “pharmaceutically-acceptable carrier” as used herein is meant one ormore compatible solid or liquid filler diluents, encapsulatingsubstances or foods or drinks, such as milk or portions thereof,including yogurt and other such foods, including, but not limited to,milk shakes and powdered milk products; non-milk products andnon-lactose containing products, including calcium carbonate, forexample. By “compatible” as used herein is meant that the components ofthe composition are capable of being comingled without interacting in amanner which would substantially decrease the pharmaceutical efficacy ofthe total composition under ordinary use situations. The pharmaceuticalcarrier in accordance with the present invention also is alsocontemplated to encompass microbial nutrients including specificprebiotics which differentially stimulate the healthy flora, and factorssuch as antimicrobial compounds, naturally occurring peptides, herbs,vitamins, minerals and plant material, which are active againsturogenital pathogens.

Some examples of substances which can serve as pharmaceutical carriersare sugars, such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethycellulose, ethylcellulose and cellulose acetates; powderedtragancanth; malt; gelatin; talc; stearic acids; magnesium stearate;calcium sulfate; vegetable oils, such as peanut oils, cotton seed oil,sesame oil, olive oil, corn oil and oil of theobroma; polyols such aspropylene glycol, glycerine, sorbitol, manitol, and polyethylene glycol;agar; alginic acids; pyrogen-free water; isotonic saline; and phosphatebuffer solution; skim milk powder; as well as other non-toxic compatiblesubstances used in pharmaceutical formulations such as Vitamin C,estrogen and echinacea, for example. Wetting agents and lubricants suchas sodium lauryl sulfate, as well as coloring agents, flavoring agents,lubricants, excipients, tabletting agents, stabilizers, anti-oxidantsand preservatives, can also be present.

Accordingly, in a preferred form of establishing, maintaining orrestoring a healthy urogenital flora, the patient is administered atherapeutically effective amount of at least one Lactobacillus iners anda pharmaceutically acceptable carrier in accordance with the presentinvention. A most preferred probiotic organism is a Lactobacillus inersY16329. Another preferred probiotic organism is L. iners CCP-1.Optionally, a second probiotic organism is administered in conjunctionwith the Lactobacillus iners. Preferably, the second probiotic organismis selected from the group comprising L. crispatus, L. jensenii, L.casei, L. salivarius, L. reuteri, L. rhamnosus, L. casei ss alactosus,L. fermentum and L. brevis. Most preferably, the second probioticorganism is either L. rhamnosus GR-1, L. fermentum B-54, L. reuteriRC-14 or L. fermentum RC-16.

Another preferred composition comprises at least one Lactobacillus inersand a prebiotic and a pharmaceutically acceptable carrier. A preferredprebiotic is inulin. Other preferred prebiotics includefructo-oligosaccharides and milk.

The introduction or administration of probiotics to pregnant women inaccordance with the present invention will provide protection againstinfections such as bacterial vaginosis, Group B streptococci, urinarytract infections and others which are capable of adversely affecting thefetus, the newborn and the mother. Accordingly, in a preferred method ofestablishing a healthy, normal urogenital flora in women before orduring pregnancy, a vaginal culture is obtained from the individual andthe culture is assayed for the presence of the lactobacilli orbifidobacteria. Selected lactobacilli or bifidobacteria are isolated,purified, grown and optionally frozen and stored (e.g., commercially)for future use by the donor. Alternatively, selected lactobacilli orbifidobacteria are orally or vaginally re-administered in atherapeutically effective amount and form to the donor. In a preferredembodiment at least one probiotic organism is isolated from a donor inneed of flora restoration or maintenance. Isolated organisms areresuspended in a pharmaceutical carrier and grown to a concentrationpermitting the reintroduction or reimplantation of about 109organisms/ml. Reimplanted probiotic organisms are preferablyadministered daily until the birth of the baby, or daily for about 52weeks in non-pregnant women. Vaginally reintroduced probiotic organismsare preferably administered once per week.

The introduction or administration of lactobacilli probiotics to theintestine and passage onto the urogenital tract, and their subsequentproduction of anti-pathogenic products (e.g., biosurfactants, acids,hydrogen peroxide, bacteriocins) modulates the immune response againstinfection and disease and reduces the risk of medical device associatedinfections. While not wishing to be bound by a particular mechanism,host responses are stimulated which inhibit pathogens and/or create amicroenvironment less conducive to pathogen spread in women.Accordingly, in a preferred embodiment of stimulating host responses, amedical device is contacted or coated with Lactobacillus at aconcentration of about 109 organisms/cm2 prior to introduction into apatient in need of such device. Medical devices contemplated by thepresent invention include but are not limited to: intrauterine devices,catheters, stents, drainage lines, pads and tampons, for example.

Although the present invention is not bound by any one theory or mode ofoperation, it is believed that, at least to some degree, a combinationof adhesion of Lactobacillus iners and the production by Lactobacillusiners of one or more inhibitory substances is responsible for excludingpathogens and/or reducing their numbers at the site of agastrointestinal or genito-urinary infection.

From the standpoint of physical exclusion, the attachment ofLactobacillus iners acts as a block to pathogens by inhibiting access toreceptor sites or interfering with the virulence of the pathogen.Although complete exclusion of pathogens theoretically can occur, themost common finding of the results of the present invention is thatthere is a reduction in pathogen numbers compared to probioticorganisms, e.g., lactobacilli or bidfidobacteria. In other words,although some probiotic organisms may not completely exclude pathogens,they are still capable of interfering with pathogen colonization invivo.

In a further aspect of the present invention, the pharmaceuticalcompositions of the present invention are employed for the treatment ofinfection. Thus, the present invention provides methods of treating aninfection in a subject by administering a therapeutically effectiveamount of a pharmaceutical composition of the present invention.

The term “therapeutically effective amount” means the dose required totreat an infection.

By “infection” is meant a pathological disorder, the onset, progressionor the persistence of the symptoms of which requires the participationof one or more Lactobacillus strains.

The term “treatment” or “treat” refers to effective inhibition,prevention or treatment of the infection.

The term “subject” refers to any mammalian subject. Preferably, thesubject is a human.

The invention is further illustrated by the following specific exampleswhich are not intended in any way to limit the scope of the invention.

EXAMPLE 1

Twenty, asymptomatic post-menopausal, otherwise healthy women wererecruited. None of the recruits were receiving estrogen, anti-microbialor any other type of prescribed therapy. Two vaginal swabs and amid-stream urine sample were collected from each of the subjects on amonthly basis for four consecutive months.

Microscope slide smears were made from vaginal swabs collected from eachindividual, Gram-stained and scored by the Nugent method (Nugent et al.(1991) J. Clin Microbiol 29:297-301). Nugent results were graded as 1-4(normal vaginal state), 5-7 (intermediate-grade vaginosis) and 8-10(high-grade bacterial vaginosis). Urine samples were routinely culturedfor bacteria in the St. Josephs Health Care London Med-CoreLaboratories, using either blood agar base (BDH, Germany) supplementedwith 5% blood or MacConkey (BDH) agar plates. Isolates were identifiedby the automated Vitek card system (Bio-Merieux Vitek, Inc., Hazelwood,Mo., USA).

Bacterial strains, representative of bacterial pathogens in theurogenital tract, used in this study are listed in Table 1. Gardnerellavaginalis was grown at 37° C. on blood agar base (BDH) supplemented with5% blood under CO2 conditions using the BBL Gas Pack® system (Becton andDickinson, Cockeysville, Md.). Other isolates were maintained with brainheart infusion agar (BBL) supplemented with 0.5% yeast extract, 1 mlvitamin K heme solution (0.5 mg/ml hemin, 0.05 mg/ml menadione) and 0.1%Tween 80 (Sigma Chem. Co, St Louis, Mo.) and were grown anaerobically at37° C.

DNA was extracted from 500 μl pure bacterial culture using InstageneMatrix (Bio-Rad Laboratories, Hercules, Calif.) according to themanufacturers instructions. Swabs were immediately placed in transportmedium (NCS Diagnostics Inc. Ontario, Canada) and taken to the lab forprocessing within three hours. Swabs were vigorously agitated in 1 ml ofphosphate-buffered saline (PBS, pH 7.5) to dislodge cells. These werepelleted by centrifugation (10 000×g, 5 minutes), washed once in the PBSand DNA was extracted as above.

Reactions were carried out in 0.2 ml tubes in a thermocycler (EppendorfMastercycler, Germany). Each PCR reaction (100 ìl) consisted of 10 ìl of10× buffer (10 mM Tris-HCl, 2.5 mM MgCl2, 50 mM KCl), 200 ìM dNTP(Roche, Germany), 2 ìl of glycerol (Sigma), 80 ìg BSA, 40 pmoles of eachprimer (GibcoBRL, Life Technologies, Gaithersburg, Md.), 5 U of DNA Taqpolymerase (PLATINUM, GibcoBRL), 10 ìl of the DNA preparation and madeup to volume with milli-Q H2O. Amplification conditions have beendescribed previously for the various primer sets and are listed in Table2.

Preparation of denaturing gradient gel electrophoresis (DGGE) wascarried out according to the manufacturer's guidelines for the D-code™Universal Detection System of Bio-Rad. A 100% solution was taken as amixture of 7 M urea and 40% formamide. The concentration ofpolyacrylamide, denaturant and tris-acetate buffer (TAE, 40 mM Tris, 20mM glacial acetic acid, 1 mM EDTA [pH 8.0]) was 8%, 30-50% and 1×,respectively. Solutions were de-gassed for at least 30 minutes beforethe addition of polymerisation agents: 55 μl of TEMED (N,N,N′,N′-tetramethylethylenediamine, Sigma) and 95 μl of 10% ammoniumpersulphate (Bio-Rad). Gels were allowed to polymerise overnight.Samples were mixed with 2× loading buffer (0.25 ml bromophenol blue [2%,Sigma], 0.25 ml xylene cyanol [2%, Sigma], 7 ml glycerol and 2.5 ml ofdH2O) and loaded into the wells. Gels were run at 130 V in 1× TAE, untilthe second dye front (xylene cyanol) approached the end of the gel.After electrophoresis, gels were removed, allowed to cool before theremoval of the glass plate sandwich, stained for 20 minutes in 5 μg/mlof ethidium bromide and de-stained for 10 minutes in 1× TAE. Gels werevisualised by ultra-violet transillumination and recorded (Polaroid 667instant film, Bedford, Mass.).

Fragments of interest were excised from DGGE gels by sterile scalpel andplaced into a single eppendorf. Gel pieces were washed once in 1×PCRbuffer and incubated in 20 μl of the same buffer overnight at 4° C. Fivemicrolitres of the buffer solution was used as template for PCRamplification. Re-amplification was conducted using either the universalbacterial or eukaryotic PCR primers as described previously, but without“GC-clamps”. Sequences of the reamplified fragments were determined bythe dideoxy chain termination method. Searching of the partial 16S and18S DNA sequences was conducted using the Genbank DNA database and theBLAST algorithm (Altschul et al. (1990) J. Mol. Biol 215:403-10).Identities of isolates were determined on the basis of the highestscore.

Results

Nugent Scores and Culture Results Of twenty non-symptomatic women testedon the first day of the study, 15 of the 20 (75%) had eitherintermediate or high-grade Nugent scores, with 8 (40%) being indicativeof BV (Table 3). Eleven of the women had intermediate or high-gradeNugent scores over the period of the entire study. Of the six women witha normal reading on the first day, four maintained this state throughoutthe study, when tested at monthly intervals (Table 3). The detection andidentification of bacterial isolates from culture varied, with therebeing little or no correlation to Nugent score and in some cases wheremolecular methods indicated that there were different types of bacteria,none were cultured (Table 3).

DGGE and PCR Identification of Bacteria Using DGGE analysis of PCRproducts from the V2-V3 region of the 16S rRNA gene, it was possible togroup many of the bacterial isolates (Table 1). The Gram-negative E.coli and the Klebsiella isolates migrated the same distance in the DGGEgel, whereas the Proteus isolate moved differently. The Gram-positiveEnterococcus faecalis strains tested migrated the same distance to thatof the type strain, where Enterococcus faecium was different. All of theStaphlococcus isolates tested migrated similarly to the S. epidermidistype strain. While only one G. vaginalis and S. agalactiae strains weretested, their DNA fragments migrated differently, compared to the otherisolates. Representative PCR amplicons of urogenital isolates were mixedand added to lanes in DGGE gels to aid in further identification of suchbacteria from vaginal samples. Species-specific PCR primers for S.agalactiae did not amplify DNA from any of the other microrganismstested. However, E. coli and G. vaginalis primers were also found toamplify DNA from other related microorganisms (Table 1).

DGGE and Sequencing of Bacterial DNA Fragments from Vaginal SamplesBacterial DGGE profiles from the vaginal samples tested were lesscomplex compared to those reported from other autochthonous populationswithin the human body, using the same PCR primers (Tannock et al. (2000)Appl Environ Microbiol 66:2578-88). Also, some of the microfloraprofiles were identical between different women (i.e. Subjects 303 and308). There was little correlation between the number of DNA fragmentsobserved in a DGGE gel from a vaginal sample and the Nugent score andculture results of the subject when tested at that time. However, womenwith Nugent scores deemed as normal, generally had few (1 to 3) dominantDNA fragments observed, and when sequenced had homology to Lactobacillusspecies (Table 4). The loss in detection of a Lactobacillus species byDGGE, observed in monthly samples, correlated to a more negative Nugentscore, where the detection of a strain after a period of absence,resulted in the reverse (Subjects 309 and 317).

Some women with intermediate-grade Nugent scores had no bacterial DNAfragments detected in DGGE gels, few bacteria were recovered by cultureand samples were mostly PCR negative for the three species tested.(Table 3, Subjects 302 [months 2 and 3], 306, 313 and 314). Samples fromwomen with asymptomatic BV (Nugent scores 8-10) often had a DNA fragmentin the DGGE gel that correlated to, and were sequence or PCR positivefor, G. vaginalis (Table 3 and 4). The microbial composition, asobserved by DGGE analysis of women with high-grade scores, varied withsome possessing single fragments (Subject 310), while others were morecomplex (Subject 316). Women with more diverse DGGE profiles did notnecessarily have a stable bacterial population and changes could bedetected between monthly samples (Subject 316), without affecting Nugentscoring (Table 3).

PCR-DGGE using Eukaryotic PCR primers Many repetitive bands occurred inall of the vaginal samples, presumably because mammalian host DNA wasbeing amplified. DNA sequencing of dominant band in the gel showed thatthe band was 100% identical to part of the human 18S rRNA gene (Genebankaccession number M10098). The other unique fragments that occurred insome of the samples could not be re-amplified after excision from thegel.

EXAMPLE 2

Nineteen premenopausal Caucasian women, with no symptoms or signs ofvaginal or urinary infection and who were otherwise healthy, wererecruited. Each signed an Informed Consent under a protocol approved bythe human ethics review board at the University of Western Ontario. Noneof the recruits were receiving anti-microbial or any other type ofprescribed therapy. None were using spermicidal products.

Deep vaginal swabs were collected by rotating throughout the vaginalvault, from each of the subjects prior to the start of the study at day0. For the ten subjects vaginally instilling lactobacilli (subjects260-269), one capsule containing 1×109 colony forming units (CFU) ofLactobacillus fermentum RC-14 and Lactobacillus rhamnosus GR-1 wasinserted daily into the vagina following the initial swab, for threedays. Additional swabs were collected from all women on days 3, 7, 14,21 and at six months in the subjects who received probiotics. Two swabswere collected per subject at each sampling point, one for the cultureof lactobacilli for RAPD analysis, the other for direct bacterial DNAextraction for PCR-DGGE. Once taken, swabs were immediately placed intransport medium (NCS Diagnostics Inc., Ontario, Canada) and taken tothe lab for processing within three hours.

Culturing and DNA Fingerprinting of Lactobacillus Strains by RAPD

Vaginal swabs were agitated in 1 ml of sterile phosphate-buffered saline(PBS, pH 7.5) and serially diluted. To determine the persistence ofstrains L. rhamnosus GR-1 and L. fermentum RC-14 within the vagina,aliquots of each dilution were plated onto MRS plates (BBL, Becton andDickinson, Cockeysville, Md.) containing fusidic acid (32 ìg/ml, SigmaChemical Co, St Louis, Mo.) and tetracycline (5 ìg/ml, Sigma),respectively, and incubated anaerobically using the BBL Gas Pack® systemat 37″-C for 48 hours. Ten repreentative colonies from each subject wereselected for testing by RAPD analysis. Colonies were grown in onemillilitre of MRS broth overnight for DNA extraction and RAPD analysisby the method of Coakley et al. (1996) J. Inst. Brew. 102:349-54, exceptfor the primers (5′-ACG AGG CAC-3′ and 5′-ACG CGC CCT-3′, GibcoBRL, LifeTechnologies, Gaithersburg, Md.) as described elsewhere. Gardiner et al.(2002) Clin Diagn Lab Immunol 9(1):92-6; Tilsala-Timisjarvi et al.(1998) Appl Environ Microbiol 64(12):4816-9. Thermocycler conditionsconsisted of; forty cycles of 94° C. for 30 seconds, annealing at 36° C.for 30 seconds and elongation at 72° C. for 2 minutes. An initialdenaturation step at 94° C. for five minutes and a final extension stepat 72° C. for 10 minutes were also included. Ten microlitres of each PCRproduct was analysed by agarose gel electrophoresis (1.5%, agarose LE,Roche, Germany, 1× tris-acetate buffer [TAE, 40 mM Tris, 20 mM glacialacetic acid, 1 mM EDTA, pH 8.0], 5 mg/ml ethidium bromide). Molecularweight marker (100 bp ladder, GibcoBRL) and RAPD-PCR products fromrepresentatives of both probiotic strains were also included in the gelfor comparison. Electrophoresis of gels was conducted at 100 V forapproximately two hours. Gels were visualised by UV-transilluminationafter ethidium bromide staining (5 μg/ml).

Extraction of Bacterial DNA from Swabs and PCR Amplification for DGGE

Swabs were vigorously agitated in 1 ml of PBS to dislodge cells. Thesewere pelleted by centrifugation (10 000×g, 5 minutes), washed once inPBS and total DNA was extracted using Instagene Matrix (Bio-RadLaboratories, Hercules, Calif.), according to the manufacturers'instructions. PCR reactions were carried out in 0.2 ml tubes in athermocycler (Eppendorf Mastercycler, Germany). The PCR primers andamplification conditions of Walter et. al. were utilised. (2000) APPEnviron Microbiol 66(1):297-33 Primer HDA1-GC (5′-CGC CCG GGG CGC GCCCCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG T-3′; the GCclamp is in boldface) and HDA2 (5′-GTA TTA CCG CGG CTG CTG GCA C-3′)were obtained from GibcoBRL. Each PCR reaction (100 ìl) consisted of 10ìl of 10× buffer (10 mM Tris-HCl, 2.5 mM MgCl2, 50 mM KCl), 200 ìM dNTP(Roche), 2 ìl of glycerol (Sigma), 80 ìg BSA, 40 pM of each primer(GibcoBRL), 5 U of DNA Taq polymerase (PLATINUM, GibcoBRL), 10 ìl of theDNA preparation and made up to volume with milli-Q distilled H2O.

DGGE, DNA Fragment Excision from Gels, Re-Amplification and Sequencing

Preparation of DGGE gel gradients and electrophoresis were carried outaccording to the manufacturers' guidelines for the D-code™ UniversalDetection System of Bio-Rad. A 100% solution was taken as a mixture of 7M urea and 40% formamide. The concentration of polyacrylamide,denaturant and TAE was 8%, 30-50% and 1×, respectively. Samples (20 ml)were mixed with an equal volume of 2× loading buffer (0.25 mlbromophenol blue [2%, Sigma], 0.25 ml xylene cyanol [2%, Sigma], 7 mlglycerol and 2.5 ml of dH2O) and loaded into the wells. Gels were run at130 V in 1×TAE, until the second dye front (xylene cyanol) approachedthe end of the gel. After electrophoresis, gels were removed, stainedfor 20 minutes in 5 μg/ml of ethidium bromide and de-stained for 10minutes in 1×TAE. Gels were visualised by ultraviolet transillumination.Fragments of interest were excised from DGGE gels by sterile scalpel andplaced into a single eppendorf. Gel pieces were washed once in 1×PCRbuffer and incubated in 20 μl of the same buffer overnight at 4° C.Five-microlitres of the buffer solution was used as template for PCRamplification. Re-amplification was conducted using the HDA primersdescribed previously, but without the “GC-clamp” on primer HDA-1.Sequences of the re-amplified fragments were determined by the dideoxychain termination method. Analysis of the partial 16S DNA sequences wasconducted using the Genbank DNA database and the BLAST algorithm.Altschul et al. (1990) J Mol Biol 215(3)-403-10. Identities of isolateswere determined on the basis of the highest score.

Results

The mean age of the 19 women in the study was 34.15 years (range, 22-47years). The mean age of the women who instilled the capsules containinglactobacilli was 35.40 years (range, 25-45 years).

DGGE and Fragment Sequencing Results Before Probiotic Use

Most of the vaginal samples from the nineteen women studied had arelatively “simple” bacteria flora, represented by one to three DNAfragments observed within a DGGE gel (FIG. 1.). Subjects 261, 264 and268 had five to ten fragments detected (FIG. 1.). When the dominantfragments from every sample were sequenced, the majority of women tested(15/19) had at least one sequence homologous to a species ofLactobacillus (Table 5). Most notably, a strain, recently described inthe vagina, namely Lactobacillus iners, was the most commonly recoveredspecies detected in 42% of the women.

Sequence analysis indicated that Gardnerella vaginalis was present insix of the study participants at day 0, and of these three women (250,267 and 268) would be characterized as having asymptomatic bacterialvaginosis by the Nugent criteria. Nugent et al. (1991) J Clin Microbiol29(2):297-301. In three subjects with G. vaginalis, other microorganismsnot previously found in the vagina, namely Arthrobacter sp., Caulobactersp. and Butyrivibrio fibrisolvens, were also detected. G. vaginaliscoexisted with Lactobacillus species in three subjects at the firstsampling (Table 5).

DGGE and Sequencing DNA Fragments Results After Probiotic Instillation

In four (21%) patients, there was no apparent major alteration to theexisting vaginal microflora regardless of whether one (FIG. 2, Subject263), or more DNA fragments were initially detected (subjects 262, 264and 269, gels not shown). Subject 260 acquired a L. crispatus strain(100%, AF257097) in addition to her original L. iners three days aftercommencing probiotic instillation (FIG. 2).

The G. vaginalis DNA fragment present in subject 261 disappearedimmediately following one lactobacilli treatment, and was only againdetected at day 21. This subject, along with 265 retained theirindigenous lactobacilli (excluding day 3, subject 261) but also acquireda Pseudomonas strain (day 3 and 7 respectively) and a Streptococcusagalactiae strain at day 7 (subject 261). At day 21 the flora returnedto the same as it had been prior to treatment in both subjects. OtherDNA fragments observed in the last two samples in the DGGE gel fromsubject 261, when sequenced, were homologous to L. iners and were likelyto be spurious PCR artefacts. Satokari et al. (2001) Appl EnvironMicrobiol 67(2):504-13; Von et al. (1997) FEMS Microbiol Rev21(3):213-29, Subject 268 had a DNA fragment of Butyrivibriofibrisolvens present at day 0, and although the intensity of thefragment significantly decreased at day 3, it was similar to the day 0microflora in subsequent day 7, 14 and 21 samples tested (FIG. 2). Thefollow up of samples from women after six months showed that most women(10/18, one non-compliant) had altered DGGE profiles, indicating thattheir microflora had changed compared to those observed at day zero.

RAPD Analysis of Lactobacillus Isolates

The presence of the instilled exogenous Lactobacillus species could notalways be detected within the vaginal samples using PCR-DGGE (FIG. 2).However, RAPD detected the exogenous lactobacilli strains in 80% womenafter one week and 20% after three weeks (FIG. 3). The detection ofinstilled Lactobacillus strains by RAPD (data not shown) inverselycorrelated with detection of G. vaginalis through DGGE and sequenceanalysis in samples from subject 261.

According to the results achieved with the present invention,Lactobacillus iners, not previously detected by others who examined thevaginal flora Antonio et al. (1999) J Infect Dis 180(6)1950-6; Giorgi etal. (1987) Microbiologica 10(4):377-84; Larsen et al. (2001) Clin InfectDis 32(4):e69-77; Reid et al. (1996) FEMS Immunol Med Microbiol15(1)23-6, is clearly a common constituent of the women sampled and isuseful in methods of restoring urogential health in women. This straindoes not grow on the major selective mediums utilised by others for theisolation of Lactobacillus, specifically MRS and Rogosa-Sharp. Falsen etal. (1999) Int J Syst Bacteriol 1:217-21. This might explain theirfailure to detect it, or it may have been confused with members of theL. acidophilus complex. Falsen et al. (1999) Int J Syst Bacteriol1:217-21; Kulen et al. (2000) J Appl Microbiol 89(3):511-6. Thediscovery highlights the advantage of using PCR-DGGE and sequencing forbacterial identification. L. iners is a previously unrecognizedprobiotic useful in protecting the vagina from disease and restoringvaginal and urogenital health.

The discovery of three strains not previously detected in the vagina wasalso achieved. Arthrobacter are Gram-positive organisms isolated fromsoil. Some members of this genus are now regarded as opportunisticpathogens, having been recovered from blood and urine. Hou et al. (1998)Int J Syst Bacteriol 2:423-9; Wauters et al. (2000) J Clin Microbiol38(6):2412-5; Funke et al. (1996) J Clin Microbiol 34(10):2356-63.Caulobacter sp. are freshwater organisms and Butyrivibrio fibrisolvensis a rumen organism known for producing conjugated linoleic acid.Although the precise origin of these organisms in the three subjects isuncertain, the findings do suggest that the vaginal microflora may alsobe influenced by environmental organisms, perhaps acquired throughbathing and exposure to the soil.

The correlation between a healthy vaginal tract, as defined by lack ofsymptoms and signs of disease and dominance of lactobacilli Nugent etal. (1991) J Clin Microbiol 29(2):297-301, supports the belief thatcommensal lactobacilli play a major role in preventing certain types ofvaginal infections. Sobel (2000) Annu Rev Med 51:349-56. In the day 0samples, three out of six subjects had G. vaginalis in conjunction witha species of Lactobacillus. Thus the presence of lactobacilli does notnecessarily have to exclude potential pathogens from the vagina.

Persistence of microorganisms below the detection threshold of DGGE wasdemonstrated by the culturing of vaginal swabs, on selective antibioticmediums, preferential for the supplanted Lactobacillus strains andtyping isolates by RAPD. For up to twenty-one days after initialinstillation, the exogenous strains could be detected in the samplesfrom some women by RAPD, but not by PCR-DGGE. In accordance with thepresent invention, it is believed that probiotic microorganisms create aslight perturbation of the microflora following which other persistantendogenous microorganisms, including lactobacilli (such as L. crispatusin subject 260) take advantage to replenish their populations.

The PCR-DGGE has provided a means to develop a new understanding of themicroorganisms within the vagina. Previous culture studies have failedto identify a number of species present, including L. iners. Theinstillation of two probiotic strains showed that non-hydrogen peroxideproducing L. rhamnosus GR-1 colonized better than the RC-14H2O2producer, emphasizing that expression of this factor alone isinsufficient for restoration of a lactobacilli-dominant flora as hasbeen previously proposed. Vallor et al. (2001) J Infect Dis184(11):1431-6. The advantage of combination therapy is that in somewomen, perhaps those lacking in H2O2 producing lactobacilli,colonization by producers is feasible, while in other women strains withadditional properties, such as the bacteriocin production of GR-1, canlead to colonization. The DGGE technique is a most useful adjunct forclinical studies of the vaginal tract. TABLE 1 PCR Results IsolateStrain Source DGGE Profile E. coli G. vaginalis ¹ S. agalactiaeGram-negative Escherichia coli ATCC 11775 Catheter Reference strain + −− Escherichia coli Lab 31 UTI Escherichia coli + − − Escherichia coliLab 67 UTI Escherichia coli + − − Escherichia coli Lab 917 UTIEscherichia coli + − − Escherichia coli Lab C1212 Escherichia coli + − −Escherichia coli Lab C1214 Escherichia coli + − − Escherichia coli LabCo 1 Faeces Escherichia coli + − − Escherichia coli Lab Hu737Escherichia coli + − − Klebseilla Lab 280 Escherichia coli + − − Proteusmirabilis Lab 28ii Kidney infection Escherichia coli + − − Gram-positiveEnterococcus faecalis ATCC 19433T Reference strain − − − Enterococcusfaecalis ATCC 23241 Kidney infection Enterococcus faecalis − − −Enterococcus faecalis ATCC 33186 Enterococcus faecalis − − −Enterococcus faecalis Lab 1131 Enterococcus faecalis − − − Enterococcusfaecalis Lab 1136 Enterococcus faecalis − − − Enterococcus faecalis ATCC19434^(T) Reference strain − − − Enterococcus faecium ATCC 14018^(T)Reference strain − − − Garnerella vaginalis Mt. Sinai Toxic shocksyndrome Staphylococcus − − Staphlococcus aureus ATCC 35984^(T) Cathetersepsis Reference strain − − − Staphlococcus epidermidis Lab 169Staphylococcus − − − Staphlococcus epidermidis Lab 3081 Staphylococcus −− − Staphlococcus epidermidis Lab 3294 Staphylococcus − − −Streptococcus agalactiae ATCC 13813^(T) Reference strain − − −¹ Bifidobacterium adolecentis ATCC 15703T, Bifidobacterium breve ATCC15700T, Bifidobacterium infantis ATCC 15697T also gave a positive resultwith G. vaginalis specific primers

TABLE 2 Oligonucleotides used for PCR. Annealing Name Target Sequence(5′-3′) Position³ temperature (° C.) HDA-1-GC Bacteria ²TCC TAC GGG339-157 56 AGG CAG GAG HDA-2 Bacteria GTA TTA CCG 518-539 56 CGG CTG CTGGCA Eco2083 E. coli GCT TGA CAC 2083-2103 57 TGA ACA TTG AG Eco2745 E.coli GCA CTT ATC 2726-2745 57 TCT TCC GCA TT Eukl427f-GC Eukaryote ¹TCTGTG ATG 1427-1453 52 CCC TTA GAT GTT CTG GG Eukl61r Eukaryote GCG GTGTGT 1616-1637 52 ACA AAG GGC AGG G Sag40 S. agalactine CGC TGA GGT 40-6160 TTG GTG TTT ACA Sag445 S. agalactine CAC TCC TAG 445-465 60 CAA CGTTCT TC V2-R1 G. vaginalis TCG TGG AGG  5-24 62 GTT CGA TTC TG V6-U2 G.vaginalis GAC CAT GCA 1026-1045 62 CCA CCT GTG AA¹“GC-clamp” = CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G²“GC-clamp” = CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAC³ E. coli 16S rDNA numbering, except for ECO 2083/2745 (23S rDNA) andEukl427f/1616r (Saccharorhyces cerevtsiae numbering)

TABLE 3 Characteristics of twenty post-menopausal women Approximatenumber of Correlation with dominant representative DGGE bacterial PCRSubject Month Nugent Urine culture results fragments isolates E. coli G.vaginalis S. agalactiae 301 1 7 Staphylococcus (1 × 10⁶ CFU/ml) 6 G.vaginalis w + + BV 2 5 1 Mixed growth (1 × 10⁶ CFU/ml) 6 G. vaginalisw + + 3 6 1 Mixed growth (1 × 10⁶ CFU/ml) 5 G. vaginalis + + w 4 8 Nosignificant growth 6 G. vaginalis w + w BV 302 1 5/6 1 No significantgrowth 1 S. agalactiae − − + 2 5/6 1 No growth 0 − − w 3 6 1 No growth 0− − + 4 5 1 No growth 3 S. agalactiae − + + 303 1 0 N No significantgrowth 1 − − − 2 1 N No significant growth 1 − − − 3 0 N No growth 1 − −− 4 0 N No growth 1 − − − 305 1 8/9 No significant growth 2 G. vaginalis− + + BV 2 2 N No significant growth 3 G. vaginalis − + − 3 5 1 Nosignificant growth 3 G. vaginalis − + − 4 0 N No significant growth 3 G.vaginalis − + − 306 1 5/6 1 No significant growth 0 − − − 2 5 1 Nogrowth 0 − − − 3 5 1 No growth 0 − − − 4 5 1 No growth 0 − − − 307 1 9No significant growth 2 − − − BV 2 4 1 Lactobacillus (5 × 10⁷ CFU/ml) 3− − − 3 6 1 No growth 3 − − − 4 1 N No growth 2 − − − 308 1 2 N Nosignificant growth 3 − − − 2 2 N E. coli (1 × 10⁷ CFU/ml) 2 E. coli − −− Lactobacillus (1 × 10⁸ CFU/ml) 3 3 N No significant growth 2 − − − 4 3N No growth 3 E. coli − − − 309 1 2 N Mixed growth (1 × 10² CFU/ml) 2 E.faecialis − − − 2 0 N Mixed growth (1 × 10⁸ CFU/ml) 1 − − − Staphyloccus(1 × 10⁷ CFU/ml) 3 6 1 E. coli (1 × 10⁷ CFU/ml) 3 E. coli, + − − E.faecialis, G. vaginalis 4 1 N No growth 2 E. faecialis, − − − E. coli310 1 9 No growth 1 G. vaginalis − − − BV 2 10 No significant growth 1G. vaginalis − − − BV 3 10 Staphylococcus (1 × 10⁷ CFU/ml) 1 G.vaginalis − − − BV 4 9 No growth 1 G. vaginalis − − − BV 311 1 5/6 1 Nosignificant growth 5 S. agalactiae, + − − E. faecalis 2 7 No growth 5 S.agalactiae, w − − BV E. faecalis 3 9 No growth 5 S. agalactiae, + − − BVE. faecalis 4 7 No significant growth 5 S. agalactiae, − − − BV E.faecalis 312 1 5/6 1 No growth 3 − − − 2 6 1 No growth 7 − − − 3 6 1 Nogrowth 7 − − − 4 7 No growth 10 w − − BV 313 1 5/6 1 No significantgrowth 0 − − − 2 5 1 No growth 0 − − − 3 5 1 No growth 0 − − − 4 5 1 Nosignificant growth 0 w − − 314 1 5/6 1 No growth 0 − − − 2 5/6 1Klebsiella (5 × 10⁷ CFU/mf) 0 − − − 3 6 1 No growth 0 − − − 4 6 1 Nogrowth 0 − − − 315 1 8 No growth 1 G. vaginalis − + − BV 2 8 No growth 6E. coli, − + − BV G. vaginalis 3 8 No growth 6 E. coli, w + − BV G.vaginalis 4 10 No growth 6 E. coli, − + − BV G. vaginalis 316 1 10Coliforms (1 × 10⁸ CFU/ml) 7 E. coli, + + − BV E. faecalis 2 10Klebsiella (1 × 10⁸ CFU/ml) 7 E. coli, + + − BV E. faecalis 3 10Klebsiella pneumoniae 7 E. coli, + + − BV (1 × 10⁸ CFU/ml) E. faecalis 410 No growth 5 G. vaginalis − + − BV 317 1 7/8 No growth 2 − + + BV 2 1N No significant growth 2 S. agalactiae − w − 3 0 N Staphylococcusdetected 2 S. agalactiae − w + 4 8 BV No growth 4 S. agalactiae − w +318 1 0 N No significant growth 1 − w − 2 1 N No significant growth 1 −w − 3 0 N Pseudomobas aerougmosa 1 G. vaginalis − w w detected 4 0 NStaphylococcus detected 1 − w − 319 1 7 BV No growth 5 S. agalactiae, −− − E. faecium 2 6/7 BV No significant growth 5 S. agalactiae, − − − E.faecium 3 5 1 No growth 5 S. agalactiae, − − − E. faecium 4 5 1 Nogrowth 5 S. agalactiae, − − − E. faecium 320 1 1 N No significant growth1 E. faecalis − − − 2 1 N Diptheroids detected 4 E. faecalis, w + − (1 ×10⁸ CFU/ml) Staphlococcus 3 1 N Staphylococcus detected 4 E. faecalis, −− − Staphlococcus 4 0 N No significant growth 3 E. faecalis, − w −Staphlococcus 321 1 0 N No significant growth 1 − − − 2 0 N No growth 1− − − 3 7 BV No significant growth 1 E. faecalis − − − 4 5/6 1Klebsiella pneumoniae 3 E. faecalis, + w + (1 × 10⁸ CFU/ml) E. coli, G.vaginalis

TABLE 4 Sequence analysis of bands excised from DGGE gels derived frombacterial 16S rDNA extracted from vaginal swabs (Day 0) Iden- Sub- Mostclosely related tity Accession ject Nugent Band bacterial sequence (%)number 301 I 1 Lactobacillus sp. 95 AF159022 (closest sp. L gasseri) 2Lactobacillus sp. 93 AF159022 (closest sp. L. gasseri) 3 Slackia exigua98 AF101240 4 Gardnerella vaginalis 98 M58744 302 I 5 Streptococcusagalactiae 99 AF015927 303 N 6 Lactobacillus iners 100 Y16329 305 BV 7Gardnerella vaginalis 92 M58744 307 BV 8 Uncultured bacterial clone 89AF203861 9 Gardnerella vaginalis 97 M58744 308 N 10 Lactobacillus sp. 98AF159022 (closest sp. L. gasseri) 309 N 11 Lactobacillus sp. 97 AY029223(closest sp. L. crispatus) 12 Lactobacillus sp. 98 AF157035 (closest sp.L. crispatus) 310 BV 13 Gardnerella vaginalis 88 M58744 311 I 14Prevotella sp 95 AF385512 15 Prevotella buccalis 96 LI6476 16 Prevotellabuccalis 96 LI6476 312 I 17 Lactobacillus sp. 98 AF157045 (closest sp.L. fermentum) 316 BV 18 Gardnerella vaginalis 98 M58744 317 BV 19Prevotella bivia 97 LI16475 318 N 20 Lactobacillus crispatus 98 AF257097319 BV 21 Bacteroides sp. 90 AF139525 22 Peptostreptococcus sp 87 D1414123 Prevotella buccalis 95 LI6476 320 N 24 Lactobacillus iners 94 Y16329321 N 25 Lactobacillus sp. 99 AF157035 (closest sp. L. crispatus)

TABLE 5 BLAST analysis of vaginal bacterial 16S rRNA sequences ofexcised fragments from DGGE gels (Day 0 samples). Most closely relatedbacterial Subject sequence % Identity Accession number 250 Gardnerellavaginalis 98 M58744 252 Lactobacillus crispatus 100 AF259097 Gardnerellavaginalis 98 M58744 253 Lactobacillus cripatus 98 AF259097 254Lactobacillus iners 100 Y16329 255 Lactobacillus crispatus 97 AF259097256 Lactobacillus crispatus 100 AF259097 Lactobacillus iners 99 Y16329257 Lactobacillus crispatus 98 AF259097 Lactobacillus iners 100 Y16329258 Streptococcus agalactiae 100 AF015927 259 Lactobacillus gasseri 100AF243165 260 Lactobacillus iners 100 Y16329 261 Lactobacillus iners 99Y16329 Arthrobacter sp. 100 AJ243423 Gardnerella vaginalis 99 M58744 262Lactobacillus iners 96 Y16329 Lactobacillus acidophilus 97 AF375937 263Lactobacillus delbruekil 97 AF375917 264 Lactobacillus iners 92 Y16329Gardnerella vaginalis 98 M58744 265 Lactobacillus crispatus 98 AF257097266 Lactobacillus iners 96 Y16329 267 Caulobacter sp. 98 M83799Gardnerella vaginalis 97 M58744 268 Butynvibrio fibrisolvens 95 AF125217Gardnerella vaginalis 97 M58744 269 Lactobacillus crispatus 99 AF257097

1. A method for establishing a healthy bacterial flora in femalesthroughout life, comprising administering a therapeutically effectiveamount of at least one Lactobacillus iners and a pharmaceuticallyacceptable carrier.
 2. The method of claim 1, wherein said bacterialflora is gastrointestinal or urogenital flora.
 3. The method of claim 1,wherein said Lactobacillus iners is Y16329.
 4. The method of claim 1further comprising a second probiotic organism.
 5. The method of claim4, wherein said second probiotic organism is a Lactobacillus selectedfrom the group consisting of L. rhamnosus, L. acidophilus, L. fermentum,L. casei, L reuteri, L. crispatus, L. plantarum, L. paracasei, L.jensenii, L. gasseri, L. cellobiosis, L. brevis, L. delbrueckii, L.helveticus, L. salivarius, L. collinoides, L. buchneri, L. rogosae, orL. bifidum.
 6. The method of claim 4, wherein said second probioticorganism is a Biridobacterum selected from the group consisting of B.bifidum, B. breve, B. adolescentis, or B. longum.
 7. The method of claim4, wherein a prebiotic is administered in conjunction with the probioticorganism.
 8. The method of claim 1, wherein said Lactobacillus iners isadministered orally or vaginally.
 9. A method for maintaining a healthyurogenital flora in females prior to, during and after pregnancy,comprising orally administering at least one Lactobacillus iners and apharmaceutically acceptable carrier.
 10. The method of claim 9 furthercomprising a second probiotic organism.
 11. The method of claim 10,wherein said second probiotic organism is a Lactobacillus selected fromthe group consisting of L. rhamnosus, L. acidophilus, L. fermentum, L.casei L. reuteri, L. crspatus, L. plantarum, L. paracasei, L. jensenii,L. gasseri, L. cellobiosis, L. brevis, L. delbrueckii, L. helveticus, L.salivarius, L. collinoides, L. buchner, L. rogosae, or L. bifidum. 12.The method of claim 9, wherein said second probiotic organism is aBifidobacterium selected from the group consisting of B. bifidum, B.breve, B. adolescentis, or B. longum.
 13. A method for restoring healthyurogenital flora in females in need thereof comprising administering atleast one Lactobacillus iners isolated from said female and apharmaceutically acceptable carrier.
 14. A method for reducing the riskof bacterial vaginosis and bacterial vaginosis pathogens, comprisingadministering a therapeutically effective amount of at least oneLactobacillus iners and a pharmaceutically acceptable carrier.
 15. Apharmaceutical composition comprising a Lactobacillus iners and apharmaceutically acceptable carrier.
 16. The method of claim 15 furthercomprising a prebiotic.
 17. The method of any of claims 7 or 16, whereinsaid prebiotic is insulin.
 18. The method of any of claims 7 or 16,wherein said prebiotic is fructo-oligosaccharides or milk.
 19. A methodfor treatment of an infection in a subject in need by administering atherapeutically effective amount of a pharmaceutical composition ofclaim 15 or 16.